Bases moleculares de la actividad señalizadora de dUTPasas

Tesis doctoral: 356 páginas[EN]The central dogma of molecular biology purposes that every gene codifies for one protein that it will be the responsible for carry out only one function. Nevertheless, the number of multiple function proteins, known as moonlighting proteins, is increasing constantly. The dUTPases are an example of these moonlighting proteins. In particular, the model of which this thesis is about focuses on the moonlighting activity of dUTPases codified by phages of Staphylococcus aureus, which are able to induce the mobility of some pathogenicity islands that are present in this organism, also known as SaPIs. The protein Stl, which is codified by the own SaPI, blocks the mobility of SaPIs. The dUTPases of certain phages of S. aureus induce the mobility of these islands through the interaction to the repressor Stl, in a case that seems to be a clear example of cell signaling mechanism. Using macromolecular X-ray crystallography, as the main technique, and also in vivo and in vitro assays, this thesis tries to deal with the molecular mechanism through dUTPases carry out this new function. The obtained data allow us to suggest a mechanistic model for this novel signaling ability for dUTPases codified by phages of S. aureus. This mechanism, which is similar to eukaryotic G protein mechanism, implies the conserved motifs present in both monomeric and trimeric dUTPases, additional no conserved motifs and the dUTP as second messenger. In addition, comparisons at sequence and structural level between these two dUTPases families allow us to suggest this mechanism as “universal mechanism” for signaling ability of these proteins in all organisms from virus to eukaryotic organisms. This thesis, in addition, deals with the signaling molecular mechanism of dimeric dUTPases codified by phages of S. aureus. This mechanism, apparently, differs with the previously described for trimeric dUTPases and leaves a lot of non resolved question relating to the manner by which these proteins carry out with this new function.[ES] El dogma central de la biología molecular propone que para cada gen se traduce en una proteína que será responsable de una única función. No obstante, cada vez son más numerosos los casos de proteínas multifunción o moonlighting proteins capaces de llevar a cabo múltiples funciones. Entre estas proteínas moonlighting encontramos las dUTPasas. En concreto, y el modelo sobre el que gira el presente trabajo de tesis, nos hemos centrado en la actividad moonlighting que presentan las dUTPasas codificadas por fagos de Staphylococcus aureus, capaces de inducir la movilidad de islas de patogenicidad presentes en el genoma de este organismo. Las islas de patogenicidad de S. aureus, más conocidas como SaPIs, se encuentra reprimidas por un inhibidor general codificado por la propia isla, la proteína Stl. Las dUTPasas de ciertos fagos de S. aureus inducen la movilidad de estas SaPIs, a través de su unión a Stl, en lo que parece ser un claro mecanismo de señalización celular. Utilizando como técnica principal la cristalografía de moléculas mediante Rayos-X, suplementada con ensayos in vivo e in vitro, esta tesis pretende abordar el mecanismo molecular mediante el cual las dUTPasas llevan a cabo esta nueva función. Los resultados obtenidos, nos han permitido proponer un modelo de mecanismo para esta novedosa capacidad señalizadora que presentan las dUTPasas de fagos de S. aureus. Este mecanismo, similar al de las proteínas G de eucariotas, implica motivos conservados en dUTPasas triméricas y monoméricas, motivos adicionales y el dUTP como segundo mensajero. Además, la comparación a nivel de secuencia y estructural entre dUTPasas de estas dos familias permite proponer este mecanismo como un mecanismo “universal” para la capacidad señalizadora de estas proteínas en organismos que van desde virus hasta eucariotas. En este trabajo se aborda también el mecanismo molecular de señalización de dUTPasas diméricas de fagos de S. aureus, un mecanismo aparentemente muy diferente para el descrito en las triméricas y que deja sin resolver muchas cuestiones en cuanto a cómo actúan estas proteínas.Para la realización de esta Tesis, Jorge Donderis Martínez ha disfrutado de una beca lanzadera del CIBER de Enfermedades Raras y un contrato en la modalidad de prácticas suscrito en el marco del Programa Junta para la Ampliación de Estudios (JAE). El trabajo se ha enmarcado en los proyectos “Estructura y función de máquinas moleculares implicadas en redes de señalización de microorganismos.” (BIO2010-15424) y “Análisis global de mecanismos reguladores antisentido en Staphylococcus aureus.” (PIM2010EPA-00606) financiados por el Ministerio de Ciencia e Innovación; y ”Estructura, función, reconocimiento y evolución en señalización celular: de lo bacteriano (sistemas de dos componentes) a lo universal (dUTPasas).” (BIO2013-42619-P) financiado por el Ministerio de Economía y Competitividad, ; de los cuales el doctorando ha estado contratado a través de los proyectos PIM2010EPA-00606 y BIO2010-15424.Peer reviewe

Bases moleculares de la actividad señalizadora de dUTPasas

El dogma central de la biología molecular propone que para cada gen se traduce en una proteína que será responsable de una única función. No obstante, cada vez son más numerosos los casos de proteínas multifunción o moonlighting proteins capaces de llevar a cabo múltiples funciones. Entre estas proteínas moonlighting encontramos las dUTPasas. En concreto, y el modelo sobre el que gira el presente trabajo de tesis, nos hemos centrado en la actividad moonlighting que presentan las dUTPasas codificadas por fagos de Staphylococcus aureus, capaces de inducir la movilidad de islas de patogenicidad presentes en el genoma de este organismo. Las islas de patogenicidad de S. aureus, más conocidas como SaPIs, se encuentra reprimidas por un inhibidor general codificado por la propia isla, la proteína Stl. Las dUTPasas de ciertos fagos de S. aureus inducen la movilidad de estas SaPIs, a través de su unión a Stl, en lo que parece ser un claro mecanismo de señalización celular. Utilizando como técnica principal la cristalografía de moléculas mediante Rayos-X, suplementada con ensayos in vivo e in vitro, esta tesis pretende abordar el mecanismo molecular mediante el cual las dUTPasas llevan a cabo esta nueva función. Los resultados obtenidos, nos han permitido proponer un modelo de mecanismo para esta novedosa capacidad señalizadora que presentan las dUTPasas de fagos de S. aureus. Este mecanismo, similar al de las proteínas G de eucariotas, implica motivos conservados en dUTPasas triméricas y monoméricas, motivos adicionales y el dUTP como segundo mensajero. Además, la comparación a nivel de secuencia y estructural entre dUTPasas de estas dos familias permite proponer este mecanismo como un mecanismo “universal” para la capacidad señalizadora de estas proteínas en organismos que van desde virus hasta eucariotas. En este trabajo se aborda también el mecanismo molecular de señalización de dUTPasas diméricas de fagos de S. aureus, un mecanismo aparentemente muy diferente para el descrito en las triméricas y que deja sin resolver muchas cuestiones en cuanto a cómo actúan estas proteínas.The central dogma of molecular biology purposes that every gene codifies for one protein that it will be the responsible for carry out only one function. Nevertheless, the number of multiple function proteins, known as moonlighting proteins, is increasing constantly. The dUTPases are an example of these moonlighting proteins. In particular, the model of which this thesis is about focuses on the moonlighting activity of dUTPases codified by phages of Staphylococcus aureus, which are able to induce the mobility of some pathogenicity islands that are present in this organism, also known as SaPIs. The protein Stl, which is codified by the own SaPI, blocks the mobility of SaPIs. The dUTPases of certain phages of S. aureus induce the mobility of these islands through the interaction to the repressor Stl, in a case that seems to be a clear example of cell signaling mechanism. Using macromolecular X-ray crystallography, as the main technique, and also in vivo and in vitro assays, this thesis tries to deal with the molecular mechanism through dUTPases carry out this new function. The obtained data allow us to suggest a mechanistic model for this novel signaling ability for dUTPases codified by phages of S. aureus. This mechanism, which is similar to eukaryotic G protein mechanism, implies the conserved motifs present in both monomeric and trimeric dUTPases, additional no conserved motifs and the dUTP as second messenger. In addition, comparisons at sequence and structural level between these two dUTPases families allow us to suggest this mechanism as “universal mechanism” for signaling ability of these proteins in all organisms from virus to eukaryotic organisms. This thesis, in addition, deals with the signaling molecular mechanism of dimeric dUTPases codified by phages of S. aureus. This mechanism, apparently, differs with the previously described for trimeric dUTPases and leaves a lot of non resolved question relating to the manner by which these proteins carry out with this new function

Virus satellites drive viral evolution and ecology

Virus satellites are widespread subcellular entities, present both in eukaryotic and in prokaryotic cells. Their modus vivendi involves parasitism of the life cycle of their inducing helper viruses, which assures their transmission to a new host. However, the evolutionary and ecological implications of satellites on helper viruses remain unclear. Here, using staphylococcal pathogenicity islands (SaPIs) as a model of virus satellites, we experimentally show that helper viruses rapidly evolve resistance to their virus satellites, preventing SaPI proliferation, and SaPIs in turn can readily evolve to overcome phage resistance. Genomic analyses of both these experimentally evolved strains as well as naturally occurring bacteriophages suggest that the SaPIs drive the coexistence of multiple alleles of the phage-coded SaPI inducing genes, as well as sometimes selecting for the absence of the SaPI depressing genes. We report similar (accidental) evolution of resistance to SaPIs in laboratory phages used for Staphylococcus aureus typing and also obtain the same qualitative results in both experimental evolution and phylogenetic studies of Enterococcus faecalis phages and their satellites viruses. In summary, our results suggest that helper and satellite viruses undergo rapid coevolution, which is likely to play a key role in the evolution and ecology of the viruses as well as their prokaryotic hosts

Laforin, a dual specificity protein phosphatase involved in Lafora disease, is phosphorylated at Ser25 by AMP-activated protein kinase

Carlos Romá-Mateo et alt.Lafora progressive myoclonus epilepsy [LD (Lafora disease)] is a fatal autosomal recessive neurodegenerative disorder caused by loss-of-function mutations in either the EPM2A gene, encoding the dual-specificity phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Previously, we and others showed that laforin and malin form a functional complex that regulates multiple aspects of glycogen metabolism, and that the interaction between laforin and malin is enhanced by conditions activating AMPK (AMP-activated protein kinase). In the present study, we demonstrate that laforin is a phosphoprotein, as indicated by two-dimensional electrophoresis, and we identify Ser25 as the residue involved in this modification. We also show that Ser25 is phosphorylated both in vitro and in vivo by AMPK. Lastly, we demonstrate that this residue plays a critical role for both the phosphatase activity and the ability of laforin to interact with itself and with previously established binding partners. The results of the present study suggest that phosphorylation of laforin-Ser25 by AMPK provides a mechanism to modulate the interaction between laforin and malin. Regulation of this complex is necessary to maintain normal glycogen metabolism. Importantly, Ser25 is mutated in some LD patients (S25P), and our results begin to elucidate the mechanism of disease in these patientsThis work was supported the Spanish Ministry of Education and Science [grant number SAF2008-01907 (to P.S.)]; the Generalitat Valenciana [grant number Prometeo 2009/051 (to P.S.)]; the National Institutes of Health [grant numbers R00NS061803, P20RR020171, R01NS070899 (to M.S.G.)]; and the University of Kentucky College of Medicine startup funds (to M.S.G.)Peer reviewe

Indigenous Ethnicity and Low Maternal Education Are Associated with Delayed Diagnosis and Mortality in Infants with Congenital Heart Defects in Panama.

This is the first study in Panama and Central America that has included indigenous populations in an assessment of the association between socioeconomic variables with delayed diagnosis and mortality due to congenital heart defects (CHD).A retrospective observational study was conducted. A sample calculation was performed and 954 infants born from 2010 to 2014 were randomly selected from clinical records of all Panamanian public health institutions with paediatric cardiologists. Critical CHD was defined according to the defects listed as targets of newborn pulse oximetry screening. Diagnoses were considered delayed when made after the third day of life for the critical CHD and after the twentieth day of life for the non-critical. A logistic regression model was performed to examine the association between socioeconomic variables and delayed diagnosis. A Cox proportional hazards model was used to assess the relationship between socioeconomic features and mortality.An increased risk of delayed diagnosis was observed in infants with indigenous ethnicity (AOR, 1.56; 95% CI, 1.03-2.37), low maternal education (AOR, 1.57; 95% CI, 1.09-2.25) and homebirth (AOR, 4.32; 95% CI, 1.63-11.48). Indigenous infants had a higher risk of dying due to CHD (HR, 1.43; 95% CI, 1.03-1.99), as did those with low maternal education (HR, 1.95; 95% CI, 1.45-2.62).Inequalities in access to health care, conditioned by unfavourable socioeconomic features, may play a key role in delayed diagnosis and mortality of CHD patients. Further studies are required to study the relationship between indigenous ethnicity and these adverse health outcomes

Another look at the mechanism involving trimeric dUTPases in Staphylococcus aureus pathogenicity island induction involves novel players in the party

13 páginas, 5 figuras, 3 tablas, material suplementario en NAR onlineWe have recently proposed that the trimeric staphylococcal phage encoded dUTPases (Duts) are signaling molecules that act analogously to eukaryotic G-proteins, using dUTP as a second messenger. To perform this regulatory role, the Duts require their characteristic extra motif VI, present in all the staphylococcal phage coded trimeric Duts, as well as the strongly conserved Dut motif V. Recently, however, an alternative model involving Duts in the transfer of the staphylococcal islands (SaPIs) has been suggested, questioning the implication of motifs V and VI. Here, using state-of the-art techniques, we have revisited the proposed models. Our results confirm that the mechanism by which the Duts derepress the SaPI cycle depends on dUTP and involves both motifs V and VI, as we have previously proposed. Surprisingly, the conserved Dut motif IV is also implicated in SaPI derepression. However, and in agreement with the proposed alternative model, the dUTP inhibits rather than inducing the process, as we had initially proposed. In summary, our results clarify, validate and establish the mechanism by which the Duts perform regulatory functions.Ministerio de Economia y Competitividad (Spain) [BIO2013-42619-P to A.M.]; Medical Research Council (UK) [MR/M003876/1]; ERC-ADG-2014 Dut-signal (from EU) [Proposal no 670932 to J.R.P]; CSIC JAE-Doc Postdoctoral contract (Programa «Junta para la Ampliación de Estudios»), European Social Fund (to E.M.); FPU13/02880 (to J.R.C.), FPI BES-2014-068617 Predoctoral Fellowships (to C.A.). Diamond Light Source block allocation group (BAG) Proposal [MX10121]; Spanish Synchrotron Radiation Facility ALBA Proposal [2014060897]; European Community's Seventh Framework Programme [FP7/2007-2013]; BioStruct-X [283570]. Funding for open access charge: Ministerio de Economia y Competitividad (Spain) [BIO2013-42619-P to A.M.]; Medical Research Council (UK) [MR/M003876/1]; ERC-ADG-2014 Dut-signal (from EU) [Proposal no 670932 to J.R.P].Peer reviewe

Virus Satellites Drive Viral Evolution and Ecology

19 páginas, 4 figuras, 4 tablasVirus satellites are widespread subcellular entities, present both in eukaryotic and in prokaryotic cells. Their modus vivendi involves parasitism of the life cycle of their inducing helper viruses, which assures their transmission to a new host. However, the evolutionary and ecological implications of satellites on helper viruses remain unclear. Here, using staphylococcal pathogenicity islands (SaPIs) as a model of virus satellites, we experimentally show that helper viruses rapidly evolve resistance to their virus satellites, preventing SaPI proliferation, and SaPIs in turn can readily evolve to overcome phage resistance. Genomic analyses of both these experimentally evolved strains as well as naturally occurring bacteriophages suggest that the SaPIs drive the coexistence of multiple alleles of the phage-coded SaPI inducing genes, as well as sometimes selecting for the absence of the SaPI depressing genes. We report similar (accidental) evolution of resistance to SaPIs in laboratory phages used for Staphylococcus aureus typing and also obtain the same qualitative results in both experimental evolution and phylogenetic studies of Enterococcus faecalis phages and their satellites viruses. In summary, our results suggest that helper and satellite viruses undergo rapid coevolution, which is likely to play a key role in the evolution and ecology of the viruses as well as their prokaryotic hostsThis work was supported by grants Consolider-Ingenio CSD2009-00006, BIO2011- 30503-C02-01, ERAnet-Pathogenomics PIM2010EPA-00606 and MR/M003876/1 from the Medical Research Council (UK) to JRP, BIO2010-15424 to AM, and BFU2012-30805 to SFE, all from the Spanish Ministerio de Economía y Competitividad (MINECO), and grant R01AI022159 to RPN and JRP, from the USA National Institutes of Health. AB acknowledges support from the Royal Society, NERC, BBSRC and AXA research fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscriptPeer reviewe

A polyamorous repressor: deciphering the evolutionary strategy used by the phage­inducible chromosomal islands to spread in nature

1 página con el abstract del póster presentado a 44th FEBS Congress Krakow, Poland. July 6-11, 2019Staphylococcus aureus pathogenicity islands (SaPIs) are a family of related 15­17Kb mobile genetic elements that carry and disseminate superantigen and other virulence genes. SaPIs reside passively in the bacterial chromosome, repressed by a master repressor called Stl, encoded by the own SaPI. The key feature of their mobility and spread is the induction by helper phages of their excision, replication, and efficient encapsidation into specific small­headed phage­like infectious particles. After infection or induction of a resident helper phage, SaPIs are de­repressed by the specific protein­protein interaction of phage proteins with Stl. SaPIs have developed a fascinating mechanism to ensure their promiscuous transfer by targeting with the Stl repressor structurally unrelated phage proteins performing the same conserved function. Combining structural biology approach and functional characterization in­vivo and in­vitro we decipher the molecular mechanism of this elegant strategy by which the SaPI hijacks the phage process to sense the starting of the lytic cycle. Our structural studies show that the Stl of the island SaPIbov1 combines a canonic HTH N­terminal domain to bind DNA, and sequentially acquires new domains which act as recognizing modules for the different phage proteins (antirepressors). Our in­vivo and in­vitro data deciphers the molecular mechanism that underlies the interaction between the Stl repressor and different phage coded antirepressors, showing how each Stl module mimics the substrate for each anti­repressor type. The interaction of Stl with different types of anti­repressor always disrupts the Stl dimer, implying the DNA dissociation and SaPI derepression. Our results establish the molecular mechanism of the interaction event that detonates the intra­ and inter­ generic transference of the clinically relevant SaPIs.Peer reviewe

Socioeconomic features of infants with congenital heart defects in Panama according to ethnicity. 2010–2014.

<p>Socioeconomic features of infants with congenital heart defects in Panama according to ethnicity. 2010–2014.</p

Survival curves for infants with congenital heart defects in Panama. 2010–2014.

<p>Survival curves for infants with congenital heart defects in Panama. 2010–2014.</p